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cDNA preparation from degraded RNA - (Sep/29/2013 )

I isolate RNA from plant via iRIS method, when i run it on denaturing gel ,i found no integral band, i made cDNA via Revet aid kit fermentas, when i checked it via PCR with House keeping gene with 50 bp ladder, an exact band of 160bp was get but i also found a band of 50bp, i dont know whether it is primer dimer or some amplified product. I also used ABS random primer kit , but got the same result. when checked in real time PCR , the Ct-value was coming at range of 25-27 which is not acceptable for house keeping, tell me what should i do.

-parul goel-

Do you see the 50bp product in your controls without primer? You have used this primer before with no additional bands or is this a recently designed primer? Does the appearance of the additional band only occur with this sample?


If you lack an intact band during the denaturing gel run, then your RNA has been degraded.  Do you see a low-molecular weight smear?  That's the telltale sign of RNA degradation.  You might want to re-isolate your RNA.